RESUMEN
Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
Asunto(s)
ADN de Cadena Simple , Edición Génica , Recombinasa Rad51 , Reparación del ADN por Recombinación , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Humanos , Edición Génica/métodos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Sistemas CRISPR-Cas , Células HEK293 , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Reparación del ADN por Unión de ExtremidadesRESUMEN
Meiotic recombination is a key process facilitating the formation of crossovers and the exchange of genetic material between homologous chromosomes in early meiosis. This involves controlled double-strand breaks (DSBs) formation catalyzed by Spo11. DSBs exhibit a preferential location in specific genomic regions referred to as hotspots, and their variability is tied to varying Spo11 activity levels. We have refined a ChIP-Seq technique, called SPO-Seq, to map Spo11-specific DSB formation in Saccharomyces cerevisiae. The chapter describes our streamlined approach and the developed bioinformatic tools for processing data and comparing with existing DSB hotspot maps. Through this combined experimental and computational approach, we aim to enhance our understanding of meiotic recombination and genetic exchange processes in budding yeast, with the potential to expand this methodology to other organisms by applying a few modifications.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Meiosis/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Biología Computacional/métodosRESUMEN
During meiosis, Spo11 generates DNA double-strand breaks to induce recombination, becoming covalently attached to the 5' ends on both sides of the break during this process. Such Spo11 "covalent complexes" are transient in wild-type cells, but accumulate in nuclease mutants unable to initiate repair. The CC-seq method presented here details how to map the location of these Spo11 complexes genome-wide with strand-specific nucleotide-resolution accuracy in synchronized Saccharomyces cerevisiae meiotic cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Análisis de Secuencia de ADN/métodos , Reparación del ADNRESUMEN
This study aimed to evaluate the influence of combined intermittent fasting (IF) and high-intensity interval training (HIIT) on morphology, caspase-independent apoptosis signaling pathway, and myostatin expression in soleus and gastrocnemius (white portion) muscles from healthy rats. Sixty-day-old male Wistar rats (n = 60) were divided into four groups: control (C), IF, high-intensity-interval training (T), and high-intensity-interval training and intermittent fasting (T-IF). The C and T groups received ad libitum chow daily; IF and T-IF received the same standard chow every other day. Animals from T and T-IF underwent a HIIT protocol five times a week for 12 weeks. IF reduced gastrocnemius mass and increased pro-apoptotic proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in soleus and cleaved-to-non-cleaved PARP-1 ratio and myostatin expression in gastrocnemius white portion. HIIT increased AIF and apoptosis repressor with caspase recruitment domain expression in soleus and cleaved-to-total PARP-1 ratio in gastrocnemius muscle white portion. The combination of IF and HIIT reduced fiber cross-sectional area in both muscles, increased EndoG and AIF expression, and decreased cleaved-to-non-cleaved PARP-1 ratio in gastrocnemius muscle white portion. Muscle responses to IF and HIIT are directly impacted by the muscle fiber type composition and are modulated, at least in part, by myostatin and caspase-independent apoptosis signaling.
Asunto(s)
Factor Inductor de la Apoptosis , Apoptosis , Ayuno , Entrenamiento de Intervalos de Alta Intensidad , Fibras Musculares de Contracción Lenta , Atrofia Muscular , Miostatina , Ratas Wistar , Transducción de Señal , Animales , Masculino , Apoptosis/fisiología , Ayuno/metabolismo , Ayuno/fisiología , Miostatina/metabolismo , Entrenamiento de Intervalos de Alta Intensidad/métodos , Ratas , Transducción de Señal/fisiología , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Factor Inductor de la Apoptosis/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Rápida/patología , Endodesoxirribonucleasas/metabolismo , Condicionamiento Físico Animal/métodos , Condicionamiento Físico Animal/fisiología , Músculo Esquelético/metabolismo , Ayuno Intermitente , Poli(ADP-Ribosa) Polimerasa-1RESUMEN
BACKGROUND: Deoxyribonuclease 2 (DNase II) plays a key role in clearing cytoplasmic double-stranded DNA (dsDNA). Deficiency of DNase II leads to DNA accumulation in the cytoplasm. Persistent dsDNA in neurons is an early pathological hallmark of senescence and neurodegenerative diseases including Alzheimer's disease (AD). However, it is not clear how DNase II and neuronal cytoplasmic dsDNA influence neuropathogenesis. Tau hyperphosphorylation is a key factor for the pathogenesis of AD. The effect of DNase II and neuronal cytoplasmic dsDNA on neuronal tau hyperphosphorylation remains unclarified. METHODS: The levels of neuronal DNase II and dsDNA in WT and Tau-P301S mice of different ages were measured by immunohistochemistry and immunolabeling, and the levels of DNase II in the plasma of AD patients were measured by ELISA. To investigate the impact of DNase II on tauopathy, the levels of phosphorylated tau, phosphokinase, phosphatase, synaptic proteins, gliosis and proinflammatory cytokines in the brains of neuronal DNase II-deficient WT mice, neuronal DNase II-deficient Tau-P301S mice and neuronal DNase II-overexpressing Tau-P301S mice were evaluated by immunolabeling, immunoblotting or ELISA. Cognitive performance was determined using the Morris water maze test, Y-maze test, novel object recognition test and open field test. RESULTS: The levels of DNase II were significantly decreased in the brains and the plasma of AD patients. DNase II also decreased age-dependently in the neurons of WT and Tau-P301S mice, along with increased dsDNA accumulation in the cytoplasm. The DNA accumulation induced by neuronal DNase II deficiency drove tau phosphorylation by upregulating cyclin-dependent-like kinase-5 (CDK5) and calcium/calmodulin activated protein kinase II (CaMKII) and downregulating phosphatase protein phosphatase 2A (PP2A). Moreover, DNase II knockdown induced and significantly exacerbated neuron loss, neuroinflammation and cognitive deficits in WT and Tau-P301S mice, respectively, while overexpression of neuronal DNase II exhibited therapeutic benefits. CONCLUSIONS: DNase II deficiency and cytoplasmic dsDNA accumulation can initiate tau phosphorylation, suggesting DNase II as a potential therapeutic target for tau-associated disorders.
Asunto(s)
Enfermedad de Alzheimer , Endodesoxirribonucleasas , Neuronas , Proteínas tau , Animales , Proteínas tau/metabolismo , Proteínas tau/genética , Fosforilación , Ratones , Neuronas/metabolismo , Neuronas/patología , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/psicología , Enfermedad de Alzheimer/patología , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/metabolismo , Ratones Transgénicos , ADN/genética , Masculino , Femenino , Encéfalo/metabolismo , Encéfalo/patología , Ratones Endogámicos C57BLRESUMEN
DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.
Asunto(s)
ADN Viral , Genoma Viral , ADN Viral/genética , ADN Viral/metabolismo , ADN Viral/química , Microscopía por Crioelectrón , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Modelos Moleculares , Empaquetamiento del ADN , Ensamble de Virus/genética , Bacteriófagos/genética , Empaquetamiento del Genoma ViralRESUMEN
The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
Asunto(s)
Acidaminococcus , Proteínas Bacterianas , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Dominio Catalítico , Microscopía por Crioelectrón , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Acidaminococcus/enzimología , Acidaminococcus/genética , Acidaminococcus/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Estructuras R-Loop/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/química , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Modelos Moleculares , Dominios Proteicos , Relación Estructura-Actividad , Unión ProteicaRESUMEN
We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes.
Asunto(s)
Arabidopsis , Roturas del ADN de Doble Cadena , Meiosis , Arabidopsis/genética , Meiosis/genética , Reparación del ADN/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromosomas de las Plantas/genética , Profase Meiótica I/genética , Intercambio Genético , Replicación del ADN/genéticaRESUMEN
The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , ADN , Humanos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , ADN/metabolismo , ADN/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Edición Génica , Endonucleasas/metabolismo , Endonucleasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Reparación del ADN por Unión de Extremidades , División del ADN , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.
Asunto(s)
Arabidopsis , Marcación de Gen , Arabidopsis/genética , Marcación de Gen/métodos , Transformación Genética , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Plantas Modificadas Genéticamente/genéticaRESUMEN
Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.
Asunto(s)
Aciclovir , Antivirales , Endodesoxirribonucleasas , Herpesvirus Humano 1 , Antivirales/farmacología , Células Vero , Chlorocebus aethiops , Animales , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Aciclovir/farmacología , Ganciclovir/farmacología , Foscarnet/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Cidofovir/farmacología , Humanos , Bromodesoxiuridina/análogos & derivadosRESUMEN
Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.
Asunto(s)
Inosina , Inosina/metabolismo , Humanos , Poli A/metabolismo , Especificidad por Sustrato , Hipoxantina/metabolismo , Hipoxantina/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/químicaRESUMEN
TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.
Asunto(s)
Proteínas Bacterianas , Proteínas Asociadas a CRISPR , Clostridium botulinum , Elementos Transponibles de ADN , Endodesoxirribonucleasas , Intrones , ARN Guía de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , Recombinación Homóloga , Empalme del ARN , ARN Guía de Sistemas CRISPR-Cas/genética , Transposasas/metabolismo , Transposasas/genética , Clostridium botulinum/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismoRESUMEN
Tailed double-stranded DNA bacteriophage employs a protein terminase motor to package their genome into a preformed protein shell-a system shared with eukaryotic dsDNA viruses such as herpesviruses. DNA packaging motor proteins represent excellent targets for antiviral therapy, with Letermovir, which binds Cytomegalovirus terminase, already licensed as an effective prophylaxis. In the realm of bacterial viruses, these DNA packaging motors comprise three protein constituents: the portal protein, small terminase and large terminase. The portal protein guards the passage of DNA into the preformed protein shell and acts as a protein interaction hub throughout viral assembly. Small terminase recognises the viral DNA and recruits large terminase, which in turn pumps DNA in an ATP-dependent manner. Large terminase also cleaves DNA at the termination of packaging. Multiple high-resolution structures of each component have been resolved for different phages, but it is only more recently that the field has moved towards cryo-EM reconstructions of protein complexes. In conjunction with highly informative single-particle studies of packaging kinetics, these structures have begun to inspire models for the packaging process and its place among other DNA machines.
Asunto(s)
ADN Viral , Proteínas Virales , ADN Viral/genética , ADN Viral/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Empaquetamiento del Genoma Viral/fisiología , Empaquetamiento del ADN , Bacteriófagos/genética , Bacteriófagos/fisiología , Bacteriófagos/metabolismo , Genoma ViralRESUMEN
A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.
Asunto(s)
Sistemas CRISPR-Cas , Dependovirus , Edición Génica , Vectores Genéticos , Edición Génica/métodos , Dependovirus/genética , Sistemas CRISPR-Cas/genética , Vectores Genéticos/genética , Animales , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismoRESUMEN
Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , ADN/metabolismo , ADN/genética , ARN/metabolismo , ARN/genética , División del ADN , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Edición Génica/métodos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Francisella/genéticaRESUMEN
Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.
Asunto(s)
Aedes , Anopheles , Sistemas CRISPR-Cas , Animales , Anopheles/genética , Aedes/genética , Variación Genética , ARN Guía de Sistemas CRISPR-Cas/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Genoma de los Insectos , Mosquitos Vectores/genética , Edición Génica , Proteínas BacterianasRESUMEN
The revolutionary technology of CRISPR/Cas has reshaped the landscape of molecular biology and molecular engineering. This tool is of interest to researchers in multiple fields, including molecular diagnostics, molecular biochemistry circuits, and information storage. As CRISPR/Cas spreads to more niche areas, new application scenarios and requirements emerge. Developing programmability and compatibility of CRISPR/Cas becomes a critical issue in the new phase. Here, we report a redundancy-based modular CRISPR/Cas12a synergistic activation platform (MCSAP). The position, length, and concentration of the redundancy in the split DNA activators can finely regulate the activity of Cas12a. With the redundant structure as an interface, MCSAP serves as a modular plug-in to seamlessly integrate with the upstream molecular network. MCSAP successfully performs three different tasks: nucleic acid detection, enzyme detection, and logic operation. MCSAP can work as an effector for different molecular networks because of its compatibility and programmability. Our platform provides powerful yet easy-to-use tools and strategies for the fields of DNA nanotechnology, molecular engineering, and molecular biology.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Proteínas Asociadas a CRISPR/metabolismo , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , ADN/genética , ADN/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , LógicaRESUMEN
In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.
Asunto(s)
Proteínas de Unión al ADN , Endodesoxirribonucleasas , Endonucleasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Endonucleasas/metabolismo , Endonucleasas/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/química , Humanos , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Modelos Moleculares , Fosforilación , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Roturas del ADN de Doble Cadena , Ácido Anhídrido Hidrolasas/metabolismo , Ácido Anhídrido Hidrolasas/genética , Mutación , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Reparación del ADN , Activación EnzimáticaRESUMEN
Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.